The present invention is directed to DNA polymerases, and more particularly, to a novel mutation of Thermus aquaticus and Thermus fiavus DNA polymerases exhibiting enhanced thermostability over any form of these enzymes now known. The invention is also directed to recombinant DNA sequences encoding such DNA polymerases, and vector plasmids and host cells suitable for the expression of these recombinant DNA sequences. The invention is also directed to a novel formulation of the DNA polymerases of the present invention and other thermostable DNA polymerases, which formulation of enzymes is capable of efficiently catalyzing the amplification by PCR (the polymerase chain reaction) of unusually long and faithful products.
DNA polymerase obtained from the hot springs bacterium Thermus aquaticus (Taq DNA polymerase) has been demonstrated to be quite useful in amplification of DNA, in DNA sequencing, and in related DNA primer extension techniques because it is thermostable. Thermostable is defined herein as having the ability to withstand temperatures up to 95.degree. C. for many minutes without becoming irreversibly denatured, and the ability to polymerize DNA at high temperatures (60.degree. to 75.degree. C.). The DNA and amino acid sequences described by Lawyer et al., J. Biol. Chem. 264:6427 (1989), GenBank Accession No. J04639, define the gene encoding Thermus aquaticus DNA polymerase and the enzyme Thermus aquaticus DNA polymerase as those terms are used in this application. The highly similar DNA polymerase (Tfl DNA polymerase) expressed by the closely related bacterium Thermus flavus is defined by the DNA and amino acid sequences described by Akhmetzjanov, A. A., and Vakhitov, V. A. (1992) Nucleic Acids Research 20:5839, GenBank Accession No. X66105. These enzymes are representative of a family of DNA polymerases, also including Thermus thermophilus DNA polymerase, which are thermostable. These enzymes lack a 3-exonuclease activity such as that which is effective for editing purposes in DNA polymerases such as E. coli DNA polymerase I, and phages T7, T3, and T4 DNA polymerases.
Gelfand et al., U.S. Pat. No. 4,889,818 describe a wild-type (abbreviation used here: WT), native Thermus aquaticus DNA polymerase. Gelland et al., U.S. Pat. No. 5,079,352 describe a recombinant DNA sequence which encodes a mutein of Thermus aquaticus DNA polymerase from which the N-terminal 289 amino acids of Thermus aquaticus DNA polymerase have been deleted (claim 3 of '352, commercial name Stoffel Fragment, abbreviation used here: ST), and a recombinant DNA sequence which encodes a mutein of Thermus aquaticus DNA polymerase from which the N-terminal 3 amino acids of Thermus aquaticus DNA polymerase have been deleted (claim 4 of '352, trade name AmpliTaq, abbreviation used here: AT). Gelland et al. report their muteins to be "fully active" in assays for DNA polymerase, but data as to their maximum thermostability is not presented.
The development of other enzymatically active mutein derivatives of Thermus aquaticus DNA polymerase is hampered, however, by the unpredictability of the impact of any particular modification on the structural and functional characteristics of the protein. Many factors, including potential disruption of critical bonding and folding patterns, must be considered in modifying an enzyme and the DNA for its expression. A significant problem associated with the creation of N-terminal deletion muteins of high-temperature Thermus aquaticus DNA polymerase is the prospect that the amino-terminus of the new protein may become wildly disordered in the higher temperature ranges, causing unfavorable interactions with the catalytic domain(s) of the protein, and resulting in denaturation. In fact, a few deletions have been constructed which appear to leave the identifiable domain for DNA polymerase intact, yet none of these deletions have thermostability at temperatures as high as 99.degree. C.
While Thermus aquaticus DNA polymerase has shown remarkable thermostability at much higher temperatures than that exhibited by other DNA polymerases, it loses enzymatic activity when exposed to temperatures above 95.degree.-97.degree. C. Moreover, its fidelity at 72.degree. C. (the recommended temperature for DNA synthesis) is limited to an effective error rate of approximately 1/9000 bp. Gelland et al.'s mutein ST of Thermus aquaticus DNA polymerase (with an N-terminal 289 a.a. deletion) is significantly more stable than AT, but ST exhibits significantly decreased activity when cycled to 98.degree. C., and much less, if any, activity when cycled to 99.degree. C., during the denaturation phase of PCR cycles.
Kainze et al. (Analytical Biochem. 202:46-49(1992) report a PCR amplification of over 10 kb: a 10.9 kb and a 15.6 kb product, utilizing an enzyme of unpublished biological source (commercially available as "Hot Tub" DNA polymerase). Kainze et al. report achieving a barely visible band at 15.6 kb after 30 cycles, starting with 1 ng of .lambda. DNA template per 100 ul of reaction volume. The efficiency of this amplification was shown to be relatively low, although a quantitative calculation of the efficiency was not presented, Attempts by Kainze et al. to make WT Thermus aquaticus DNA polymerase perform in the 10-15 kb size range were not successful, nor have successful results been reported by anyone else for any form of Thermus aquaticus DNA polymerase in this size range. There is no report of any longer DNA products amplifiable by PCR.
A DNA polymerase which retains its thermostability at 98.degree. or 99.degree. C. would allow more efficient and convenient DNA analysis in several situations including "colony PCR" (see FIG. 5), and/or allow thermal cycler overshoot without inactivation of the enzyme activity. A thermostable DNA polymerase or DNA polymerase formulation which exhibits improved fidelity relative to AT or WT Thermus aquaticus DNA polymerase at optimum temperatures for synthesis would be highly desirable for applications in which the target and product DNA is to be expressed rather than merely detected. The PCR amplification method is currently limited by two factors: The length of the products obtainable, and the fidelity of those products. A thermo stable DNA polymerase preparation capable of efficient amplification of DNA spans in excess of 6 kb would significantly expand the scope of applications of PCR. For instance, whole plasmids, and constructs the size of whole plasmids, could be prepared with this method, which would be especially valuable in cases in which a portion of the DNA in question is toxic or incompatible with plasmid replication when introduced into E. coli. If this thermostable DNA polymerase preparation simultaneously conferred increased fidelity to the PCR amplification, the resulting large products would be much more accurate, active and/or valuable in research and applications, especially in situations involving expression of the amplified sequence. If the thermostable DNA polymerase preparation allowed, in addition, more highly concentrated yields of pure product, this would enhance the method of PCR to the point where it could be used more effectively to replace plasmid replication as a means to produce desired DNA fragments in quantity.